One of the main goals of developmental biology is to elucidate the molecular mechanisms that govern the generation of distinct cell types from unspecified precursors. A detailed knowledge of these mechanisms will not only help us understand fundamental principles of normal ontogeny but also explain, and ultimately correct, instances where development has derailed and disease has resulted. The basic-helix-loop-helix-zipper transcription factor Mitf is encoded by a gene whose mutations in birds, rodents, or man are associated with abnormalities in the formation of neural crest-derived and neuroepithelial-derived melanocytes and may lead to skin pigmentation defects, sensorineural deafness, and eye pathology including retinal degeneration. Our recent work indicated that Mitf mutations lead to an early arrest in the development of neural crest-derived melanoblasts which are resistant to growth factor stimulation and lack expression of the Kit tyrosine kinase receptor. In contrast, mutant retinal pigment epithelium (RPE) cells hyperproliferate, lack expression of melanocyte-specific genes, and eventually differentiate into a second stratified retina. This differential effect on the two pigment cell populations is likely due to differences in transcriptional target genes. To identify candidate target genes, RT-PCR/ microarray analyses are being performed using wild type and mutant neural crest cultures as well as microdissected wild type and mutant RPE. Since activation of different target genes may result from the action of different Mitf isoforms and/or cofactors present in the different lineages, we are identifying such isoforms and cofactors using the yeast-two-hybrid system and are expressing them in appropriate transgenic models. Also, using dissociated neural crest and organotypic optic vesicle cultures, we are identifying extracellular factors which are involved in turning on Mitf expression and thus in determining the respective melanoblast lineages. Each of these approaches takes into account the availability of a vast array of mutations of the murine Mitf gene that affect either its different promoter regions or its common coding region. The ultimate goal of these studies is to characterize the network of factors involved in the generation and function of melanocytes which are evidently of crucial importance for the development and function of mammalian sensory organs.